Leukocyte esterase detection from throat swab

ABSTRACT

The present invention relates to the field of infectious diseases. The invention specifically relates to the diagnostic test for acute bacterial pharyngitis. The test is used in screening for Group A Beta Haemolytic  Streptococcus  by the identifying the presence of leukocyte esterase in the throat. The Leukocyte Esterase Throat Swab Test is compared to the Rapid Step Test for efficiency in terms of fast delivery of results.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Indian Patent Application No.33/CHE/2015, filed Jan. 2, 2015, the content of which is incorporated byreference herein in its entirety.

FIELD OF INVENTION

The present invention relates diagnostic aid in the field of infectiousdiseases. The invention specifically relates to the diagnostic test foracute bacterial pharyngitis. The test is used in screening for Group ABeta Haemolytic Streptococcus by the identifying the presence ofleukocyte esterase in the throat.

BACKGROUND OF INVENTION

Pharyngitis is the inflammation of the pharynx, a region in the back ofthe throat. In most cases it is quite painful, and it is the most commoncause of a sore throat. Most of the acute cases are caused by viralinfections (40-80%), with the remainder being caused by bacterialinfections, fungal infections, or irritants such as pollutants orchemical substances.

Amongst the bacterial organisms that cause the acute pharyngitis, themost common are Group A beta-haemolytic Streptococci. The other types ofbacteria causing this disease are non-Group A beta-haemolyticstreptococci and Fusobacterium, though less common.

The bacterial pharyngitis caused by streptococci will be calledstreptococcal pharyngitis here onwards. The typical symptoms ofstreptococcal pharyngitis are soar throat, fever with temperature above38° C., tonsillar exudates, and enlarged tender cervical lymph nodes.The minor symptoms could be headache, nausea, vomiting, abdominal pain,muscle ache, scarlantiniform rash and petechiae on the palate. It iscontagious by means of contact.

Though symptoms like red eyes, hoarseness, runny nose or mouth ulcersare the first leads for the diagnosis of streptococcal pharyngitis, theyare not conclusive, more so in the absence of fever. The confirmatorytests can be either throat culture or the Rapid Strep Test. Throatculture involves taking a swab of the infected area or the exudates andculturing the sample on a suitable culture medium. This test is morereliable, specific for Group A beta-haemolytic Streptococci with highsensitivity and is affordable. The Rapid Strep test or Rapid AntigenDetection Test works by detecting the presence of GAS (Group AStreptococci) in the throat of a patient by responding to GAS-specificantigens on a throat swab. When the sample is applied on an antigenincorporated film strip, it changes color if the test is positive. Itgives the results within several minutes.

There is a downside to both these testing procedures. In case of thethroat culture, the results take at least 48 hours to be revealed and aculture requires special facilities. This will delay the administrationof necessary medication given to the patient that will avoid furthercomplications like the rheumatic fever, and local suppurativecomplications. In the case of Rapid Strep Test, the downside is the highcost of the film strips that contain the antibodies for GAS antigenincorporated in them. Although, rapid strep test cannot distinguish GASinfection from asymptomatic carriage of the said organism, it issufficient to provide immediate symptomatic treatment.

In an article published by Shaikh et al. in Pediatrics 2012 March,160(3): 487-493, titled ‘Accuracy and Precision of the Signs & Symptomsof Strep. Pharyngitis in children—a Systematic Review’, it was statedthat no individual symptoms or signs were effective in confirmingstreptococcal pharyngitis. It was suggested that the symptoms and signs,either individually or combined into predictive indicators, cannot beused to definitely diagnose or rule out streptococcal pharyngitis.

In another article published by Maltezou H C et al. in J. Antimicrob.Chemother. 2008 December 62(6):1407-12 dol: 1093/jac/dkn376.Epub 2008sept 11, titled ‘Evaluation of rapid antigen detection test in thediagnosis of streptococcal pharyngitis in children and its impact onantibiotic prescription’, the usefulness of Rapid Antigen Detection Test(RADT) in the diagnosis of streptococcal pharyngitis in children and itsimpact on antibiotic prescription was stated to be studied and that itwas found that RADT provided appropriate guidance to treat strep throatinfections and reduce the need for unnecessary cultures and usage ofantibiotics.

In an article published by Forward K V et al. in Canadian J. Infect.dis. Med. Microbiology 2006, Jul. 17(4)221-3, titled ‘A comparisonbetween the strep A Rapid Test device and conventional culture for thediagnosis of strep A Pharyngitis’, stated that it was found that incases of strep pharyngitis the rapid strep test has a sensitivity ofonly 72%.

In the Text book of Pediatrics, 13th edition—Streptococci pages 577-578,authored by Richard Behrman et al., the lesions caused by thestreptococcal pharyngitis were described to be characterized by edema,hyperemia and infiltration of polymorphonuclear leukocytes.

Joshi D et al. have disclosed in their article titled ‘Diagnosticaccuracy of reagent strip to determine CSF chemistry & cellularity’published in J. Neuroscience Rural Practice 2013 Apr. 4(2):140-5. Dol:10 4103/0976-3147.112737, the usage of urine reagent strip forsemi-quantitative assessment of protein, glucose and presence of LE inCSF thus, suggesting that the existing reagent strip can be used todiagnose meningitis in low resource settings.

Farahmed. F et al. disclosed in their publication titled ‘Diagnosis ofspontaneous bacterial peritonitis in children by reagent strip’ inActamed Iran, 20B, March 16; 51(2):125-8, their study on theeffectiveness of dipstick (LE & nitrite) for diagnosis of bacterialperitonitis in cirrhotic patients. It was found that the sensitivity,specificity, positive and negative predictive value of LE reagent mightprove as a rapid bedside diagnostic test for diagnosis of spontaneousbacterial peritonitis in cirrhotic patients.

Kelly and her colleagues, according to their article titled ‘LE in rapiddiagnosis of pediatric septic arthritis and found that LE test is veryuseful for rapid diagnosis of septic arthritis’ published in MedHypothesis 2013, February 80(2):19-3 dol 10 1016/May 2012 11026 EPub2012 Dec. 19, studied the usage of LE in rapid diagnosis of septicarthritis and found that LE test is very useful for rapid diagnosis ofseptic arthritis.

The ESCMID Sore throat Guideline Group consisting of Pelucchi. C et al.,in their article titled ‘Guideline for the management of acute sorethroat’ published in J. Clin. Microbiol. Infect. 2012 Apr. 18,supplement 1:1-28, dol:10.1111/j.1469-0691.2012.03766, outlined theguidelines to diagnose and treat patients with a strep throat. It wassuggested that the Centor Scoring should be used along with RapidAntigen Test, and recommended that antibiotics may not be used forCentor Scores of 0-2 as suppurative complications were infrequent withthese scores.

From an extensive study and understanding of the prior art, it was foundthat the throat culture, though the best and specific method, is a veryslow process to confirm the streptococcal infection. The Rapid StrepTest presents an alternative to the throat culture for rapid resultswhich can be an important strategy to reduce unnecessary antibiotic useor the delay in the treatment, thus avoiding further complications.However, this test can be costly and may not be affordable in lowresource settings.

It was also found in the prior art that early polymorphonuclearleukocyte response in the pharyngo-tonsilar region which results in therelease of leukocyte esterase in the lesions in the throat is caused bythe streptococcal pharyngitis.

The present invention presents a possible alternative method to theRapid Strep Test that can be performed at a lower cost but with equalefficiency to the Rapid Strep Test in terms of fast delivery of results.The present invention is based on the fact that the streptococcuspharyngitis causes lesions that result in the release of leukocyteesterase.

Objective of the Invention

The objective of the present invention is to provide a method ofdiagnostic test for screening for a bacterial source in acute bacterialpharyngitis infection.

Another objective of the present invention is to provide a method ofapplying the Leukocyte Esterase Test to screen for a bacterial source ofinfection in acute bacterial pharyngitis.

Yet another objective of the present invention is to show that theefficiency of the Leukocyte Esterase Test is equal to the Rapid StrepTest for a bacterial source of infection in acute bacterial pharyngitis.

It is also the objective of the present invention to show that theLeukocyte Esterase Test is more cost-effective than the Rapid StrepTest.

SUMMARY OF THE INVENTION

The present invention relates to a method of diagnostic test using theLeukocyte Esterase swab to screen for a bacterial source of infection inacute bacterial pharyngitis and also to show the similarity in theefficiency of this test to that of Rapid Strep Test in terms of fastdelivery of results.

One embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis.

One embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip which consists of a leukocytearea specific for granulocyte esterase.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip which consists of a leukocytearea specific for granulocyte esterase, comprising of the followingsteps:

-   -   i. Checking for preliminary clinical symptoms of acute bacterial        pharyngitis    -   ii. Collecting the sample from the throat with a swab    -   iii. Smearing the swab on the multi-stick test strip    -   iv. Observing for any color change.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip which consists of a leukocytearea specific for granulocyte esterase, comprising of the followingsteps:

-   -   i. Checking for preliminary clinical symptoms of acute bacterial        pharyngitis such as soar throat, fever, erythema of pharynx,        tonsils, exudates    -   ii. Collecting the sample from the pharyngo-tonsillar region of        the throat with a swab    -   iii. Smearing the swab on the leukocyte testing area which is        specific for granulocyte esterase of the multi-stick test strip    -   iv. Observing for any color change to purple color.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip which consists of a leukocytearea comprising of the following steps:

-   -   i. Checking for preliminary clinical symptoms of acute bacterial        pharyngitis such as soar throat, fever, erythema of pharynx,        tonsils, exudates    -   ii. Collecting the sample from the pharyngo-tonsillar region of        the throat with a swab    -   iii. Smearing the swab on the leukocyte esterase testing area        which is specific for granulocyte esterase of the multi-stick        test strip    -   iv. Observing for a color change to purple color wherein, the        sensitivity of the test is equal to or more than 15        wbcs/micromol³.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection followedby confirming the cause as Group A beta-haemolytic streptococcalinfection in acute bacterial pharyngitis.

Another embodiment of the present invention relates to a method ofdiagnostic test to screen for a bacterial source of infection using theLeukocyte Esterase Test followed by confirming the cause as Group Abeta-haemolytic streptococcal infection with a throat culture of thethroat sample.

Yet another embodiment of the present invention discloses that theLeukocyte Esterase Throat Swab Test is as efficient as Rapid Strep Testin terms of fast delivery of results.

Yet another embodiment of the present invention discloses that thecost-effectiveness of the Leukocyte Esterase Throat Swab Test is morethan the Rapid Strep Test.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: shows a graph drawn depicting the percentages of various signsand symptoms identified in acute bacterial pharyngitis.

FIG. 2: shows a graph drawn depicting comparative study of the resultsof leukocyte esterase throat swab test and rapid strep test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a method of diagnostic test for thescreening for a bacterial source of infection in acute bacterialpharyngitis using the Leukocyte Esterase Test from a throat swab samplewith a multi-stick test strip which consists of a leukocyte areaspecific for granulocyte esterase.

In one embodiment of the present invention the method of performing thetest for the screening for a bacterial source of infection in acutebacterial pharyngitis using the Leukocyte Esterase Test from a throatswab sample with a multi-stick test strip which consists of a leukocytearea specific for granulocyte esterase is described in the followingsteps:

-   -   i. Checking for clinical symptoms of acute bacterial pharyngitis        such as soar throat, fever, erythema of pharynx, tonsils,        exudates    -   ii. Collecting the sample from the pharyngo-tonsillar region of        the throat with a swab    -   iii. Smearing the swab on the leukocyte esterase testing area        which is specific for granulocyte esterase of the multi-stick        test strip    -   iv. Observing for a color change to purple color.

In another embodiment of the present invention the sensitivity of theLeukocyte Esterase Test from a throat swab sample with a multi-sticktest strip is equal to or more than 15 wbcs/micromol³.

In yet another embodiment of the present invention, the experimentalprocedure for comparing the efficiency of the Leukocyte Esterase Testfrom a throat swab sample with a multi-stick test strip to that of theefficiency of the Rapid Strep Test was described comprising of thefollowing steps:

A) SELECTION OF SYMPTOMS

The symptoms presented basic grounds for selection of the subjects.These symptoms can range from soar throat and fever as the preliminarycomplaints to clinically diagnosed symptoms like erythema of pharynx,tonsils, exudates, erythema of tonsils, tender anterior cervical lymphnodes, absence of cough, petechiae/haemorrhages over the palate,abdominal pain, vomiting and skin rash.

B) SELECTION OF SUBJECTS

There were 100 subjects enrolled in total each of who was of age lessthan 15. All the subjects showed at least three of the symptoms listedabove. The percentage of signs and symptoms observed in the subjects isdepicted in the table below:

TABLE 1 Other Erythema Absence signs/symptoms No. of Sore Over tonsilsPalatal Tender Ant. Cerv. Of Abd. like skin rash, Subjects Fever Throat&/or Pharynx Exudates Hemorrhage Lymph Nodes Cough Pain Vomiting 100 100100 100 20 8 85 86 6 10

C) SELECTION OF MULTI-STICK TEST STRIP

The multi-stick test strip is structured such that it consists of aleukocyte area onto which the throat swab sample is applied. Thepresence of granulocyte esterases is revealed by change of color topurple color. The esterase cleaves a derevitalized pyrazole amino acidester to liberate derevitalized hydroxyl pyrazole. This pyrazole thenreacts with diazonium salt to produce a purple color.

D) TESTING PROCEDURE

The test procedure can be explained in the following steps:

-   -   i. All the patients were first checked for the presence of signs        and symptoms listed above.    -   ii. An informed consent was taken from the parents of the        subjects, the subjects themselves being children. The assent of        the subjects was also taken.    -   iii. Three swabs of the sample were collected from the        pharyngo-tonsilar region of the throat of the subjects.    -   iv. The first swab was used for culturing the sample collected,        the second swab was used for the Rapid Strep Test, and the third        swab was used for the Leukocyte Esterase Test.

E) PRECAUTIONS TAKEN DURING THE TEST

There were some precautions taken during the test to maximize theaccuracy of the tests. None of the patients were given any antibiotic onthe first day till the culture reports were obtained. Instead, they wereasked to use saline gargles and analgesics. None of the patientsdeveloped any suppurative complications like peritonsilar abscess. Alsothe test procedure was approved by the IRB and was performed withinformed consent of the parents.

F) RESULTS

The results obtained of the tests for screening for a bacterial sourceof infection in acute bacterial pharyngitis are described below:

-   -   i. 84 out of the 100 Rapid Strep Tests done were negative.    -   ii. 16 out of the 100 Rapid Strep Tests were positive.    -   iii. 80 out of the 100 Leukocyte Esterase Tests were negative.    -   iv. 20 out of the 100 Leukocyte Esterase Tests were positive.    -   v. 9 out of the 100 cultures were positive for Group A        beta-haemolytic streptococcus.    -   vi. 9 out of the 16 positive Rapid Strep Tests were confirmed by        the throat culture test.    -   vii. 9 out of the 20 positive Leukocyte Esterase Tests were        confirmed by the throat culture test.    -   viii. Further, it was noticed that 7 out of the 20 subjects who        had exudates showed throat culture positive for streptococcus.    -   ix. Further, 6 out of the 8 subjects who had palatal        haemorrhages showed throat culture positive for streptococcus.

G) CENTOR CRITERIA

The modified Centor Criteria was used for the experimental procedure ofthe present invention for the evaluation and management of streptococcuspharyngitis. The Centor criteria method involves assignment of one pointto each criterion to decide the treatment mode of the effected subjects.The criteria are:

-   -   i. Absence of cough    -   ii. Swollen and tender cervical lymph nodes.    -   iii. Temperature above 38° C.    -   iv. Tonsilar exudate or swelling    -   v. Age less than 15 years (3 to 14 years).

Any subject positive for throat culture and satisfying the criteria wasrecalled back for treatment with antibiotics.

The results disclosed above have been tabulated in Table 2 as follows:

TABLE 2 THROAT RAPID LEUKOCYTE CULTURES STREP TEST ESTERASE TEST  9positives 16 (+)ve 20 (+)ve 91 negatives 84 (−)ve 80 (−)ve

H) ANALYSIS OF RESULTS

Chi-Square 2×2 method with two tailed p-value calculations and 3×2Fisher's Exact Test with one tailed p-value calculations were used forthe analysis of the results obtained for both the Rapid Strep Test andthe Leukocyte Esterase Test. The result of the analysis is disclosed inTable 3 below:

TABLE 3 LEUKOCYTE RAPID STREP TEST ESTERASE TEST Two tailed p-value0.0012 0.0047 One tailed p-value <0.0001 <0.0001

The sensitivities of the three tests have also been disclosed to be

-   -   Throat culture: sensitivity is 100% whereas specificity is 100%        with a positive predictive value of 100% and a negative        predictive value of 100%.    -   Rapid strep test: sensitivity is 100% whereas specificity is        92.31% with a positive predictive value of 56.25% and a negative        predictive value of 100%.    -   LE test-throat: sensitivity is 100% whereas specificity is        87.91% with a positive predictive value of 45% and a negative        predictive value of 100%.

I) COMPARISON OF PRICES

A comparison of the prices of the Rapid Strep Test and the LeukocyteEsterase Test was done and it was found that the Rapid Strep Test stripwas approximately 4 to 5$ each while the Leukocyte Esterase Testmulti-stick test strip was less than 10 cents each.

J) CONCLUSIONS

From the results of the experimental studies to compare the Rapid StrepTest and the Leukocyte Esterase Test described above, it was concludedthat:

-   -   i. The time taken for obtaining the results of Leukocyte        esterase test is almost same as that of the rapid strep test        which is about a few minutes.    -   ii. The Leukocyte Esterase Test from a throat swab sample with a        multi-stick test strip is as efficient as the Rapid Strep Test        in terms of fast delivery of results.    -   iii. The cost of a Leukocyte Esterase Test multi-stick test        strip is much less than a Rapid Strep Test strip.    -   iv. The cost-effectiveness of the Leukocyte Esterase Test is        better than the Rapid Strep Test.    -   v. Hence it can be concluded that the usefulness of the        Leukocyte Esterase Test from a throat swab sample with a        multi-stick test strip is equal to, if not better than the Rapid        Strep Test in screening for bacterial infections causing acute        pharyngitis in children.

1-9. (canceled)
 10. A method of testing a patient with bacterialpharyngitis for infection, comprising: collecting a sample from thepharyngo-tonsillar region of a throat of the patient; obtaining a resultof a leukocyte esterase test of the sample; and diagnosing the patienthas having or not having the infection.
 11. The method of claim 10,wherein the infection is a Group A beta-haemolytic streptococcalinfection.
 12. The method of claim 11, wherein the leukocyte esterasetest is a test strip.
 13. The method of claim 12, wherein the test stripincludes a leukocyte area specific for granulocyte esterase.
 14. Themethod of claim 13, wherein collecting a sample comprises swabbing witha swab.
 15. The method of claim 14, further comprising: checking forpreliminary clinical symptoms of acute bacterial pharyngitis; smearingthe swab on the test strip; and observing for any color change.
 16. Themethod of claim 15, wherein: checking for preliminary clinical symptomsof acute bacterial pharyngitis includes checking for one of sore throat,fever, erythema of pharynx, tonsils, and exudates; smearing the swab onthe test strip comprises smearing the swab on the leukocyte areaspecific for granulocyte esterase; and observing for any color changecomprises observing for any color change to purple.
 17. The method ofclaim 16, wherein the sensitivity of the test is equal to or more than15 wbcs/micromol³.
 18. The method of claim 10, further comprisingperforming the leukocyte esterase test.
 19. The method of claim 18,wherein the infection is a Group A beta-haemolytic streptococcalinfection.
 20. The method of claim 19, wherein the leukocyte esterasetest is a test strip.
 21. The method of claim 20, wherein the test stripincludes a leukocyte area specific for granulocyte esterase.
 22. Themethod of claim 21, wherein collecting a sample comprises swabbing witha swab.
 23. The method of claim 18, wherein collecting the samplecomprises swabbing the throat with a swab.
 24. The method of claim 23,wherein performing the leukocyte esterase test comprises smearing theswab on a leukocyte esterase test strip.
 25. The method of claim 24,wherein the leukocyte esterase test strip comprises a leukocyte areaspecific for granulocyte esterase.
 26. The method of claim 25, whereinsmearing the swab on the leukocyte esterase test strip comprisessmearing the swab on the leukocyte area specific for granulocyteesterase.
 27. A method of testing a patient with bacterial pharyngitisfor infection, comprising: receiving a sample taken from thepharyngo-tonsillar region of a throat of the patient; performing a testfor the infection of the throat using a leukocyte esterase test.
 28. Themethod of claim 27, wherein the leukocyte esterase test is a test strip.29. The method of claim 28, wherein the test strip includes a leukocytearea specific for granulocyte esterase.
 30. the method of claim 29,wherein the sample includes a swab and performing the test for theinfection comprises smearing the swab on the leukocyte area specific forgranulocyte esterase.